PROJECT SUMMARY The thyrotropin receptor (TSHR) plays a vital role in thyroid physiology and pathophysiology. Physiologically, the TSHR is a partner in the thyroid hormone servo-feedback loop on pituitary TSH secretion. In Graves' Disease thyroid-stimulating autoantibodies (TSAb) activate the TSHR and cause hyperthyroidism. Understanding how TSAb and TSH interact with the TSHR extracellular domain and how the TSHR structure predisposes to TSAb generation are important clinical goals. The TSHR, like the other glycoprotein hormone receptors, is a member of the G protein-coupled receptor superfamily with a very large ectodomain consistent with the large size of its ligand, TSH. The TSHR receptor ectodomain comprise three components:- 7 A cysteine-rich N-terminal domain (NTD; amino acid residues 22-41 after deletion of the signal peptide). Although containing two anti-parallel leucine-rich repeats, it has some distinctive features that permits it to be considered separately from the following component. 7 A leucine-rich repeat domain (LRD) forming a slightly curved tubular structure. Each repeat has a beta-strand on its concave surface that forms part of the TSH binding region. 7 A hinge region (approximately amino acid residues 270-412) linking the LRD to the insertion of the ectodomain into the plasma membrane. The hinge region is also unique among the glycoprotein hormone receptors in containing a ~50 amino acid segment that is deleted during intramolecular cleavage into disulfide-linked A- and B-subunits. Most is known about the TSHR LRD, whose crystal structure has been determined. However the structure of its NTD component has not been fully defined. We will continue our studies on the TSHR NTD based on our evidence that it plays a role in TSAb function, and also possibly in the generation of TSAb leading to Graves' Disease. The structure of the hinge region is entirely unknown yet is vital to understanding TSHR structure and function. Although neglected by most investigators, there is evidence that the TSHR hinge region contains part of the TSH binding site, couples TSH binding with signal transduction and influences ligand-independent constitutive activity. The hinge must also stabilize the position of the LRD relative to the membrane spanning TMD. The proposed studies on the least well understood TSHR NTD and hinge regions will provide novel insight into the structure and function of the TSH holoreceptor and its activation by TSH and TSAb in Graves' Disease.